Functional machineries to prevent and repair DNA damages are required for maintaining genome integrity and preventing tumourigenesis. A set of proteins belonging to the RecQ family is among the cancer suppressors linked to repairing lesions caused by DNA damaging agents, such as ionizing radiation and cisplatin. During the course of evolution, RecQ genes appear to have been amplified and diverged from a single copy of the RecQ gene in bacteria and yeast to five RecQ homologs in humans. The clinical phenotypes caused by mutations in different RECQ proteins in humans and mouse models indicate that they have important and non-overlapping roles in maintaining genomic integrity and cancer prevention. Since RECQ helicases are highly interactive proteins and form large protein complexes in vivo, the unique functions of each of the RECQ proteins are likely defined and regulated by the specific protein-protein interactions they form in cells. To understand how the mammalian RECQ proteins act as tumor suppressors and caretakers for our genome, it is crucial to understand the unique functions of each of the RECQ protein complexes. Recently, our lab has several breakthroughs in the study of human RECQ4 helicase. First, we successfully reported ATP-dependent RECQ4 helicase activity in vitro. Surprisingly, domain analyses uncovered two distinct ATP binding and DNA unwinding activities within the RECQ4 protein. Secondly, we report the identification of a highly purified chromatin bound RECQ4 complex from human cell extracts. We found that essential replisome factors MCM10, MCM2-7 helicase, CDC45 and GINS are the primary interaction partner proteins of human RECQ4. Moreover, circadian proteins, TIMELESS and TIPIN important for cohesion establishment and DNA replication progression are part of the RECQ4 complex. Importantly, complex formation and the association of RECQ4 with the replication origin are cell cycle regulated. Our studies allow us to conclude that RECQ4 is an integral component of the MCM replicative helicase complex participating in DNA replication in human cells. Based on our work, the goal of this proposal is to define the exact function of RECQ4 complex in DNA replication, cohesion establishment and cancer avoidance. The Specific Aims are: (1) dissecting the role of human RECQ4 in DNA replication initiation; (2) Establishing the functional relationship between RECQ4 and TIM-TIPIN heterodimer in replication fork progression, genotoxic stress response and cohesion establishment; and (3) Analysis of the cancer-associated RECQ4 (c.1390+2delT) mutation. PUBLIC HEALTH RELEVANCE: DNA replication is essential for cell growth, and abnormal DNA synthesis can result in genome instability, mutagenesis and tumorigenesis. Therefore, elucidation of the mechanisms that regulate DNA replication and the molecular functions of the involved proteins, such as RECQ4, are fundamental to our understanding of the cancer development. Crucially, our detailed knowledge on the DNA replication factors will aid in the discovery of new cancer therapy treatments, since inhibiting DNA replication factors effectively prevents cell proliferation.